Detection of glucose-derived d- and l-lactate in cancer cells by the use of a chiral NMR shift reagent

Eul Hyun Suh,Ralph J Deberardinis,Brandon Faubert,Carlos F G C Geraldes,A Dean Sherry,Sara Chirayil,Raul Ayala

doi:10.1186/s40170-021-00267-4

Abstract

BackgroundExcessive lactate production, a hallmark of cancer, is largely formed by the reduction of pyruvate via lactate dehydrogenase (LDH) to l-lactate. Although d-lactate can also be produced from glucose via the methylglyoxal pathway in small amounts, less is known about the amount of d-lactate produced in cancer cells. Since the stereoisomers of lactate cannot be distinguished by conventional 1H NMR spectroscopy, a chiral NMR shift reagent was used to fully resolve the 1H NMR resonances of d- and l-lactate.MethodsThe production of l-lactate from glucose and d-lactate from methylglyoxal was first demonstrated in freshly isolated red blood cells using the chiral NMR shift reagent, YbDO3A-trisamide. Then, two different cell lines with high GLO1 expression (H1648 and H 1395) were selected from a panel of over 80 well-characterized human NSCLC cell lines, grown to confluence in standard tissue culture media, washed with phosphate-buffered saline, and exposed to glucose in a buffer for 4 h. After 4 h, a small volume of extracellular fluid was collected and mixed with YbDO3A-trisamide for analysis by 1H NMR spectroscopy.ResultsA suspension of freshly isolated red blood cells exposed to 5mM glucose produced l-lactate as expected but very little d-lactate. To evaluate the utility of the chiral NMR shift reagent, methylglyoxal was then added to red cells along with glucose to stimulate the production of d-lactate via the glyoxalate pathway. In this case, both d-lactate and l-lactate were produced and their NMR chemical shifts assigned. NSCLC cell lines with different expression levels of GLO1 produced both l- and d-lactate after incubation with glucose and glutamine alone. A GLO1-deleted parental cell line (3553T3) showed no production of d-lactate from glucose while re-expression of GLO1 resulted in higher production of d-lactate.ConclusionsThe shift-reagent-aided NMR technique demonstrates that d-lactate is produced from glucose in NSCLC cells via the methylglyoxal pathway. The biological role of d-lactate is uncertain but a convenient method for monitoring d-lactate production could provide new insights into the biological roles of d- versus l-lactate in cancer metabolism.

Highlights

Excessive lactate production, a hallmark of cancer, is largely formed by the reduction of pyruvate via lactate dehydrogenase (LDH) to L-lactate
It is widely accepted that glucose is the predominant source of energy, glucose oxidation occurs indirectly, with astrocytes taking up glucose from the blood, converting it to lactate via glycolysis, exporting lactate for oxidation by neurons [3, 4]
We introduce here a simple Nuclear magnetic resonance (NMR) method for resolving the resonances of D- and L-lactate in any biological sample by the addition of a chiral shift reagent (SR)

Summary

Introduction

A hallmark of cancer, is largely formed by the reduction of pyruvate via lactate dehydrogenase (LDH) to L-lactate. It is widely accepted that glucose is the predominant source of energy, glucose oxidation occurs indirectly, with astrocytes taking up glucose from the blood, converting it to lactate via glycolysis, exporting lactate for oxidation by neurons [3, 4]. This means that lactate must be converted back to pyruvate by neuronal lactate dehydrogenase (LDH) before being completely oxidized in the TCA cycle. The amount of ATP generated from these extra reducing equivalents in neurons depends upon whether the lactate is converted to pyruvate via cytosolic LDH or, as has been reported in some tissues, via mitochondrial LDH [6,7,8]

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